Project Abstract White adipose tissue (WAT) is the main energy storage depot in vertebrates and tightly controls glucose homeostasis in mammals. The gut microbiota is now recognized as a central regulator of WAT in healthy organisms and significantly contributes to WAT dysfunction during obesity. Yet how the gut microbiota regulates WAT functions remains largely unknown. This proposal is focused on defining the mechanistic basis by which the microbiota controls host metabolism through the regulation of WAT functions. For the first time, we have now revealed that a highly conserved family of microRNAs acts as a central controller of WAT functions in response to microbiota-derived signals. In this proposal, we aim to dissect the mechanisms by which the microbiota regulates host metabolism and WAT functions through the modulation of this miRNA family, which will reveal critical new insights into the interactions between the microbiota and host metabolism. It is well-established that the gut microbiota controls WAT functions that determine fat mass and insulin sensitivity in healthy organisms, and dysregulation of these processes leads to the development of obesity and insulin resistance (IR), which affect millions of people worldwide. However, the microbial-derived signals and the molecular mechanisms by which the gut microbiota regulates WAT functions in health and disease are largely unknown. Excitingly, we have discovered that the microbiota induces the expression of a highly conserved family of miRNAs (miR-181) specifically in white adipocytes, to regulate energy expenditure and insulin sensitivity. Moreover, our findings reveal that dysregulation of the gut microbiota-miR-181 axis is critical for the development of obesity and IR in mice. Thus, I hypothesize that modulation of miR-181 levels in white adipocytes by the microbiota represents a central mechanism by which commensal microorganisms control host metabolism, and that its dysregulation leads to obesity. In search of the microbiota-derived signal regulating miR-181 in white adipocytes, we have discovered that circulating microbiota-derived metabolites are significantly dysregulated in obese mice and humans, including a potential negative regulator of miR-181, indole. Yet still, how the microbiota regulates miR-181 levels in WAT and how this miRNA family controls WAT functions in response to microbial signals is unknown. I propose to address this gap in knowledge in the following aims: Aim 1 will determine if indole regulates miR-181 expression in WAT adipocytes to control the progression of obesity and IR. Aim 2 will elucidate the mechanisms by which miR-181 controls white adipose functions. These studies will shed light on the mechanisms by which the expression of a critical family of microRNAs is tuned in response to dietary and environmental changes, and how these changes alter the progression of obesity and IR. As our preliminary data shows that indole and MIR-181 levels are dysregulated in obese human individuals, MIR-181 may represent a potential therapeutic target to modulate WAT function in the context of obesity.